The therapy of many diseases in mammals is limited by the absence of specific medicaments.
In infections caused by intracellular pathogens infection persists because of the insufficiency of immune response which would recognize and eliminate infected cells. Many pathogens reduce the surface expression of molecules such as the major histocompatibility complex of class I (class I MHC) in the cells invaded by said pathogens, thereby reducing the capacity of the immune system to elicit a cytolytic immune response which is elicited when T lymphocytes of the CD8+ lineage recognize and are activated by class I MHC presenting pathogen-derived epitopes. An alternative strategy by which cytolytic lymphocytes could eliminate cells invaded by a pathogen would be much desirable. Such a strategy has been proposed (EP 2 059 256) in which class II restricted epitopes derived from intracellular pathogens and coupled to a thiol-oxidoreductase motif are used to elicit cytolytic CD4+ T cells which induce apoptosis of the antigen-presenting cell (APC) presenting the cognate epitope. However, the recruitment and activation of an alternative subset of cytolytic T cells would represent a distinct possibility to increase elimination of cells infected with an intracellular pathogen.
In autoimmune diseases, as in immune responses to administration of an allofactor and in allergic diseases, it is advantageous to eliminate cells presenting peptides from an autoantigen, an allofactor or an allergen, so as to prevent any unwanted immune responses and thereby diseases associated with such unwanted immune responses. Under such circumstance epitopes from autoantigens, allofactors or allergens are primarily presented by class II MHC and the complex formed between the epitope and class II determinants activated T lymphocytes of the CD4+ lineage. This results in activation of B lymphocytes and production of antibodies to said autoantigens, allofactors or allergens. A method which would result in eliminating of APC by cytolysis would prevent CD4+ T cell activation and thereby the production of antibodies. Such a strategy has been proposed and described in patent application WO 2008/017517 A1 in which class II restricted epitopes of autoantigens or allergens, or of allofactors, respectively, are used attached to a thiol-oxidoreductase motif. Cytolytic class II-restricted CD4+ T cells elicited by exposure to class II restricted epitopes coupled to said motif induce apoptosis of APC presenting the cognate epitope. However, the recruitment and activation of alternative cytolytic T cells would represent a valuable alternative strategy.
In the case of tumors, cells escape elimination by down-regulating surface expression of class I and class II MHC determinants. Any strategy by which cytolytic T cells specific to tumor antigens would be elicited would therefore represent a much desirable strategy for the treatment of tumors. WO 2009/101205 teaches that cytolytic T cells activated by class II restricted presentation of tumor derived antigens is of use for tumor elimination. However, this approach is limited by the poor expression of MHC class II determinants by tumors.
In graft rejection, the process of chronic rejection is driven by the indirect presentation of antigens shed by the graft and presented by the recipient antigen-presenting cells to his/her own T lymphocytes. The indirect presentation occurs by presentation of graft derived epitopes by both class I and class II epitopes. T lymphocytes of the CD8 lineage activated by class I MHC presentation of graft antigens migrate to the graft wherein they mediate rejection by recognition of their cognate epitopes directly on grafted cells. Yet activation of CD8 cells require help from CD4 cells activated by indirect presentation of graft derived antigens by class II MHC determinants. WO 2009/100505 teaches that the use of class II restricted T cell epitopes derived from the graft and coupled to a thiol-oxidoreductase motif allows elimination by apoptosis of APC participating in indirect presentation. However, an alternative strategy by which another subset of cytolytic T cells would be generated would be much desirable.
Likewise, novel therapeutic approaches such as gene therapy and gene vaccination are severely limited by the host immune response to viral vectors used for transgenesis or vaccination. In both these situations, antigens derived from viral vectors are shed by cells transduced with the vector and presented to host lymphocytes by host APC, namely by indirect antigen presentation. To note is the fact that many viral vectors activate not only the adaptive immune system, leading to the production of specific antibodies and specific T cell activation, but said viral vectors also activate the innate immune system. Activation of innate immunity serves as an adjuvant for the adaptive response. WO 2009/101204 teaches that class II restricted epitopes derived from viral vectors and coupled to a thiol-oxidoreductase motif can elicit the activation of cytolytic class II restricted CD4 T cells. However, an alternative strategy is highly desirable, which would suppress activation of the innate immune system.
In all examples enumerated herein, it is obvious for the one skilled in the art that alternative strategies by which antigen-specific cytolytic T cells could be elicited, which would eliminate in an antigen-specific manner APC presenting said specific antigen, would be of much value.
The present invention presents such an alternative strategy.
Natural killer T (NKT) cells constitute a distinct subset of non-conventional T lymphocytes that recognize antigens presented by the non-classical MHC complex molecule CD1d. Two subsets of NKT cells are presently described. Type 1 NKT cells, also called invariant NKT cells (iNKT), are the most abundant. They are characterized by the presence of an alpha-beta T cell receptor (TCR) made of an invariant alpha chain, Valpha14 in the mouse and Valpha24 in humans. This alpha chain is associated to a variable though limited number of beta chains. Type 2 NKT cells have an alpha-beta TCR but with a polymorphic alpha chain. However, it is apparent that other subsets of NKT cells exist, the phenotype of which is still incompletely defined, but which share the characteristics of being activated by glycolipids presented in the context of the CD1d molecule.
NKT cells typically express a combination of natural killer (NK) cell receptor, including NKG2D and NK1.1. NKT cells are part of the innate immune system, which can be distinguished from the adaptive immune system by the fact that they do not require expansion before acquiring full effector capacity. Most of their mediators are preformed and do not require transcription. NKT cells have been shown to be major participants in the immune response against intracellular pathogens and tumor rejection. Their role in the control of autoimmune diseases and of transplantation rejection is also advocated.
The recognition unit, the CD1d molecule, has a structure closely resembling that of the MHC class I molecule, including the presence of beta-2 microglobulin. It is characterized by a deep cleft bordered by two alpha chains and containing highly hydrophobic residues, which accepts lipid chains. The cleft is open at both extremities, allowing to accommodate longer chains. The canonical ligand for CD1d is the synthetic alpha galactosylceramide (alpha GalCer). However, many natural alternative ligands have been described, including glyco- and phospholipids, the natural lipid sulfatide found in myelin, microbial phosphoinositol mannoside and alpha-glucuronosylceramide. The present consensus in the art (see reviews, such as Matsuda et al, Current Opinion in Immunology 2008, 20:358-368 and Godfrey et al, Nature reviews Immunology 2010, 11: 197-206) is that CD1d binds only ligands containing lipid chains, or in general a common structure made of a lipid tail which is buried into CD1d and a sugar residue head group that protrudes out of CD1d.
Peptides are not deemed to be able to activate NKT cells through presentation by CD1d. It was, however, suggested that long hydrophobic peptides containing bulky aminoacid residues could bind to CD1d (Castano et al, Science 1995, 269: 223-226). Observations carried out using phage display libraries expressing random sequence peptides with no defined physiological relevance, allowed establishing a theoretical consensus motif (Castano et al, Science 1995, 269: 223-226 and see below).
In fact, Castano et al showed that the cells which are activated are CD8+ T cells, namely MHC class I restricted cells, and not NKT cells. These findings teach the one skilled in the art that there is no evidence that hydrophobic peptides are presented by CD1d molecules. The physiological relevance of the claims made by Castano et al was further questioned due to the inability to elicit NKT cells under conventional immunization protocols (Matsuda et al, Current Opinion in Immunology 2008, 20:358-368 and Brutkiewicz Journal of Immunology 2006, 177: 769-775). Artificial systems such as immunization with cells transfected to overexpress CD1d and loaded in vitro with an ovalbumin-derived peptide were able to elicit NKT cells. Likewise, intradermal immunization with plasmid DNA together with murine CD1d and costimulatory molecules induce cytolytic CD1d-restricted T cells (Lee et al, Journal of Experimental Medicine 1998, 187: 433-438). Hydrophobic peptides containing a structural motif made of an aromatic residue in position P1 and P7, which represent anchoring residues for binding to CD1d hydrophobic pockets located at each end of the CD1d molecule and an aliphatic chain in position P4 were claimed by Castano et al (Science 269: 223, 1995) to contain a core motif for CD1d binding epitopes. As described above, the conclusions reached by Castano et al are not supported by data.
We made the unexpected finding that peptides encompassing an hydrophobic aminoacid sequence are in fact capable of eliciting activation of NKT cells. An example of such sequence is represented by the motif [FW]-xx-[ILM]-xx-[FW], wherein [FW] is an aminoacid selected from phenylalanine or tryptophan, and [ILM] is an aminoacid selected from isoleucine, leucine or methionine. [FW] in P7 is said to be permissive, meaning that T or H can substitute either for F or W.
We further discovered that a CD1d binding motif was particularly efficient in modulating NKT activity when coupled to a thiol-oxidoreductase motif. This motif presents a general structure of C-XX-C in which C is cysteine and X is any aminoacid except tyrosine, phenylalanine and tryptophan. Patent application WO 2008/017517 A1 teaches that class II restricted T cell epitopes coupled to a thiol-oxidoreductase motif acquire the property of transforming the phenotype and the function of class II restricted CD4 T cells into potent cytolytic cells, inducing apoptosis of APC. This effect is due to increased synapse formation between APC and T cells, a consequence of the reduction and isomerization of the CD4 molecule at the surface of T cells.
A large majority of NKT cells carry the CD4 co-receptor, the role of which remains ill defined. A recent publication, however, suggested that CD4 binds to the CD1d molecule much in the same way as its binding to WIC class II (Thedrez et al Blood 110: 251-258, 2007). In addition the presence of CD4 was shown to be required for full activation of NKT cells.
The present invention therefore relates to the use of hydrophobic peptides having the capacity to bind to CD1d and thereby recruit and activate NKT cells, coupled to a thiol-oxidoreductase motif. Such peptides ensure antigen-specificity and represent a valuable approach for the treatment of
(1) infectious diseases with intracellular pathogens, in which infected cells present hydrophobic peptides derived from the pathogen and bound to CD1d. Increased NKT recruitment and/or activity of such NKT cells would therefore concur to the elimination of infected cells;
(2) autoimmune diseases, immune responses to administration of an allofactor and allergic diseases, in which antigens associated to each of these 3 types of diseases generate hydrophobic peptides presented by CD1d. Increased recruitment and/or activity of antigen-specific NKT cells could therefore help in eliminating antigen-presenting cells and thereby eliminate an unwanted immune response;
(3) tumors, as tumor cells often express CD1d carrying tumor-specific antigens, which can be recognized by NKT cells. Increasing the activity and recruitment of such NKT cells would lead to increased tumor elimination;
(4) graft rejection, as host antigen-presenting cells present hydrophobic peptides derived from the graft in the context of CD1d. Recognition of these peptides by host NKT cells would lead to elimination of the antigen-presenting cells and abort the chronic graft rejection process;
(5) gene therapy and gene vaccination, wherein antigens from viral vectors and shed by transduced cells are presented by CD1d determinants. Recruitment and activation of NKT cells eliminating host APC through recognition of viral vector antigens would be beneficial both for persistence of transgene expression and maintenance of full immunogenicity of the transgene in gene vaccination.
In addition to the therapeutic interest of the present invention, we made the unexpected observation that addition of an oxidoreductase motif within flanking residues of CD1d epitopes increases TCR binding, which results in a much improved detection of CD4+ NKT cells. Peptides encompassing natural CD1d-restricted epitopes and at least one thioreductase motif of the CxxC format, in which C stands for cysteine and x for any aminoacids except cysteine or bulky residues, as described in the present invention, have therefore a major interest for:
(1) analytical purposes: detection of NKT cell precursor frequency before vaccination, evaluation of peptide binding affinity for CD1d complexes, follow-up of specific NKT cells during the course of vaccination or under immunosuppression, identification of cells regardless of their biological activity, identification of cells implicated in the mechanism of disease, depletion of specific NKT cells, and detection of NKT cells in situ, such in organ biopsies;
(2) preparative purposes: preparation of specific NKT cells for evaluation of function and preparation of NKT cells for culture and purification;
(3) quality control for cell population aimed at cell therapy;
(4) therapeutic purposes, including depletion of specific CD4+ NKT cells before organ grafting.